Background: Biosurfactants are biological compounds that possess many pharmacological proprieties. Among them, surfactin is one of the most active against pathogens of medical interest, such as fungi, bacteria, and enveloped viruses. Objectives: In this study, we aim to evaluate the antibacterial anti-Candida, and anti-dengue potential of a surfactin biosynthesized by Bacillus subtilis ATCC 6633. In addition, the fractional inhibitory concentration (FIC) was calculated to determine the behavior of this biosurfactant in association with antibacterial and antifungal drugs of clinical use. Materials and Methods: B. subtilis ATCC 6633 culture was maintained on nutrient agar plates, and biosurfactant production was carried out in mineral salt medium with 2% glucose. The isolated and purified surfactin was used for determination of antibacterial and antifungal effects by the broth microdilution method. A checkerboard assay was performed to determine the potential synergic effect of surfactin with gentamicin, penicillin, and nystatin. Finally, the activity against Dengue virus (DENV) was evaluated through quantification of cell viability after viral infection. Results: Although it had low cytotoxicity (CC50 >400 μg/mL), surfactin was inactive against Gram-negative and Gram-positive bacteria, Candida species, and DENV at the highest concentration tested (500 μg/mL). According to FIC values, none of the antimicrobials tested showed a synergistic association with surfactin. Conclusions: Surfactin produced by B. subtilis ATCC 6633 is not a promising antimicrobial agent, and its combination with clinically available antibiotics does not lead to a synergic effect.