Owing to the significant contribution of three-dimensional (3D) field-based QSAR toward hit optimization and accurately predicting the activities of small molecules, herein, the 3D-QSAR, in vitro antimicrobial, molecular docking, and pharmacophore modeling studies of all the isolated (R/S)-2-thioxo-DHPM-5-carboxanilides exhibiting antimicrobial activity were carried out. The screening process was performed using 46 compounds, and the best-scoring model with the top statistical values was considered for bacterial and fungal targets Bacillus subtilis and Candida albicans. As a result of 3D-QSAR analysis, compound 4v-(S)- and 4v-(R)-isomers were found to be more potent compared to the standard drugs tetracycline and fluconazole, respectively. Furthermore, the enantiomerically pure isomers 4q, 4d', 4n, 4f', 4v, 4q', 4c, and 4p' were found to be more potent than tetracycline and fluconazole to inhibit the bacterial and fungal growth against B. subtilis, Salinivibrio proteolyticus, C. albicans, and Aspergillus niger, respectively. Molecular docking analysis shows that with the glide score of -10.261 kcal/mol, 4v-(R)-isomer was found to be more potent against the fungal target C. albicans and may target the 14-α demethylase than fluconazole. Furthermore, all compounds' antiproliferative activity results showed that 4o' exhibited GI50 values between 8.8 and 34 μM against six solid tumor cell lines. Following the greater potential of 4o' toward the HeLa cell line, its kinetics study and live cell imaging were carried out. These outcomes highlight the acceptance and safety as well as the potential of compounds as effective antiproliferative and antifungal agents.