Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) derivatives possess cancerostatic and anti-infective properties thus making them candidates of possible antibacterial agents. ²-lactam antibiotics are vital weapons for the treatment of bacterial infections, but their existence and effectiveness has been faced with resistance from ²-lactamases. Therefore, the need for new effective antimicrobial drugs is very crucial. In this work, we synthesized in high yields, PBD analogs 1−3, 5 and 7−9 in three to four synthetic steps from commercially available L-proline and isatoic anhydride. MTT Assay was employed to test the in vitro cytotoxicity of PBD analogs 1, 2, 5 and 7 on cancer cell lines including MCF-7, SKBR-3, SKMEL-2, CaCo 2 and Mia Paca. These compounds decreased the cell viability of MCF-7 by roughly 20% however, 1 and 5 had no effect on the SKMEL-2 cell lines. The inhibitory efficacy of these PBDs were also tested against TEM-1 and P99 Serine class A and C ²-lactamases.