Background Biofilm is one of the causes of antibiotic resistance. One of the biofilm-producing bacteria is Staphylococcus epidermidis which has been proven to infect long-term users of urinary catheters and implant devices. The 1-monolaurin compound has been known to have an antimicrobial effect. However, its effect on clinical isolates of S. epidermidis in producing biofilm has not been established. This study was conducted to investigate the effect of 1-monolaurin towards biofilm forming clinical isolates of S. epidermidis. Methods The experiment used micro broth dilution technique which consists of test group (1-monolaurin), positive control group (rifampicin), solvent group, negative control group (clinical isolate of S. epidermidis), and media group (TSB media). The Minimal Inhibition Concentration (MIC) was determined by incubating bacteria added with 1-monolaurin (1000–1953 μg/mL) or rifampicin (250–0,488 μg/mL) for 24 h. The MIC was determined visually. After that, the incubated bacteria was cultured in TSA media to determine Minimal Bactericidal Concentration (MBC). The assessment of Biofilm inhibitory Concentration (BIC) and Biofilm Eradication Concentration (BEC) was conducted with the same way, the difference was BIC intervened directly with compound meanwhile BEC was incubated for 24 h in 37 °C before the intervention. Then, the specimen was reincubated to grow biofilm at the microplate, washed with PBS and stained with 1% of crystal violet. The optical density (OD) was measured at a wavelength of 595 nm. The percentage of BIC and BEC then were calculated, continued to probit analysis regression to determine the BIC50, BIC80, BEC50, and BEC80. Results The MIC dan MBC of 1-monolaurin and rifampicin were > 1000 μg/mL, > 1000 μg/mL, ≤0.488 μg/mL, and 1.953 μg/mL respectively. BIC50 and BIC80 of 1-monolaurin and rifampicin were 26.669 μg/mL, 168.688 μg/mL, 0.079 μg/mL, and 0.974 μg/mL respectively. The BEC50 and BEC80 of 1-monolaurin and rifampicin were 322.504 μg/mL, 1338.681 μg/mL, 5.547 μg/mL, dan 17.910 μg/mL respectively. Conclusion The 1-monolaurin can inhibit growth and eradicate the biofilm formed by clinical isolates of S. epidermidis, however, it has neither inhibit nor kill planktonic cells of S. epidermidis.