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Oleaginous fungi natively accumulate large amounts of triacylglycerides (TAG), widely used as precursors for sustainable biodiesel production. However, little attention has been paid to the diversity and roles of fungal mixed microbial cultures (MMCs) in sequencing batch reactors (SBR). In this study, a lipid-rich stream produced in the fish-canning industry was used as a substrate in two laboratory-scale SBRs operated under the feast/famine (F/F) regime to enrich microorganisms with high TAG-storage ability, under two different concentrations of NaCl (SBR-N: 0.5 g/L; SBR-S: 10 g/L). The size of the fungal community in the enriched activated sludge (EAS) was analyzed using 18S rRNA-based qPCR, and the fungal community structure was determined by Illumina sequencing. The different selective pressures (feeding strategy and control of pH) implemented in the enrichment SBRs throughout operation increased the abundance of total fungi. In general, there was an enrichment of genera previously identified as TAG-accumulating fungi (Apiotrichum , Candida , Cutaneotrichosporon , Geotrichum , Haglerozyma , Metarhizium , Mortierella , Saccharomycopsis , and Yarrowia) in both SBRs. However, the observed increase of their relative abundances throughout operation was not significantly linked to a higher TAG accumulation. [Display omitted] • Fungal diversity was explored in SBRs aiming at TAG-accumulation from waste fish oil. • A double-growth limitation strategy increased absolute fungal abundance. • Fungi with the potential for TAG-accumulation were abundant at 0.5 and 10 g/L NaCl. • TAG-accumulating genera were enriched at both salinities at the end of the operation. • Apiotrichum and Candida are promising candidates to accumulate TAG from waste fish oil. [ABSTRACT FROM AUTHOR]