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We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC 50(VeroE6)  = 2.3 μM; IC 50(Calu3)  = 0.24 μM), 3'-O-4-oxatetradodecanoyl (4b) (IC 50(VeroE6)  = 2.0 μM; IC 50(Calu3)  = 0.18 μM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC 50(VeroE6)  = 2.4 μM; IC 50(Calu3)  = 0.25 μM) derivatives exhibited less activity than RDV (IC 50(VeroE6)  = 0.85 μM; IC 50(Calu3)  = 0.06 μM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC 50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV., Competing Interests: Declaration of competing interest The increasing prevalence of COVID-19 is a severe public health problem affecting people globally. COVID-19 is taking a devastating toll on human lives. Several existing drugs and potential drug candidates such as remdesivir (RDV) have been considered for repurposing as COVID-19 treatments. Our group has previously studied the impact of fatty acylation on the antiviral activity of different anti-HIV (human immunodeficiency virus) nucleoside drugs. The conjugates were more potent and less toxic than their parent nucleoside analogs, providing a much higher selectivity index. Furthermore, the conjugates significantly enhanced the cellular uptake versus the parent analogs and corresponding physical mixtures of fatty acids and nucleosides. Herein, we report the synthesis of fatty acyl derivatives of RDV and structural characterization of their antiviral activity against SARS-CoV-2 in VeroE6 cells and Calu3 cells. Five fatty acids, myristic acid, 12-azidododecanoic acid, 12-thioethyldodecanoic acid, 4-oxatetradecanoic acid, and palmitic acid were conjugated to RDV at its 2′-O- and 3′-O-position. Most monofatty acyl derivatives of RDV demonstrated IC(50) values against SARS-CoV-2 that were only slightly decreased relative to RDV. Against the EBOV trVLP and infectious EBOV, any of the monofatty acyl conjugates had IC(50)s similar to that of RDV. Overall, these data together indicate that RDV can be modified with fatty acids at positions 2′ or 3′, without demonstrating a significant loss of antiviral activity in cell culture against SARS-CoV-2 and EBOV. Future studies will determine if these modifications result in greater long-acting effect, bioavailability, and stability in animal models., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)