We have described the synthesis of β-amino alcohol based β-lactam–isatin chimeras along with their in vitro evaluation against Trichomonas vaginalis. SAR studies showed dependence of activity on concentration as well as substituent on N-1 of β-lactam and C-5 of isatin with preference for a chloro-substituent for good activity. Although the synthesized conjugates are not as active as the standard drug metronidazole, most of the compounds showed potent activity against the G3 strain of T. vaginalis. The compound 7h having a chloro-substituent on N-aryl of the β-lactam ring as well as at the C-5 position of isatin proved to be the most active chimeric scaffold among the test series with an IC50 of 9.73 μM. The most potent compound 7h has been further evaluated for its cytotoxicity profiles against HeLa cells and the results of percent viability are compared with standard drugs bleomycin and doxorubicin. Mammalian cell cytotoxicity was comparable to bleomycin, with approximately 30% viability. The low cytotoxicity of scaffold 7h further confirms the significance of this study towards the development of novel β-lactam–isatin hybrids as new anti-trichomonas agents.