The present study investigates the antimicrobial potential of the crude extracts of two fungal strains isolated from the Vellar estuary water against clinical human pathogens. The two potent fungal isolates were identified as Fusarium oxysporum RAS 2 and Aspergillus niger RAS 3 through 18S rRNA sequencing. 5 ml of n-butyl alcohol (nBA) and ethyl acetate (EA) crude extracts confirmed significant antimicrobial properties exhibited a wide range of zone of inhibitions from 10 to 38 mm against bacterial and fungal pathogens which were collected from Government Medical College Hospital, Chidambaram, when compare to control measures. The EA and NA extract of F. oxysporum RAS 2 exhibited the highest activity (30 ± 0.7 mm) and (25 ± 0.5 mm) against Shigella sp., and E. coli, respectively. Both extracts of A. niger RAS 3 showed the highest activity (38 ± 0.8 mm) and 27 ± 0.5 mm against B. subtilis. In antifungal activity, the ethyl acetate extract of F. oxysporum RAS 2 and A. niger RAS 3 exhibited the highest activity against A. niger. The n-butyl alcohol extract of F. oxysporum RAS 2 showed the highest activity (35 ± 0.7 mm) against C. albicans, while A. niger RAS 3 demonstrated significant activity (36 ± 0.8 mm) against A. niger. FTIR investigation of the EA extracts revealed the existence of an additional ester functional group at the range  1735, which correlated with enhanced antimicrobial activity compared to other extracts lacking this group. Further characterization of these metabolites is necessary to confirm their antimicrobial properties. This study concludes that fungi from the Vellar estuary produce compounds with antimicrobial activity, making them a promising source to develop novel natural antimicrobial metabolites.