In our ongoing efforts to develop potent antitubercular agents based on the 6-phenylnicotinohydrazide, herein we report the design, synthesis and biological evaluation of three sets of 6-phenylnicotinohydrazide derivatives 8a – g , 12 and 16a , b . The designed compounds were synthesized and in vitro evaluated for their antitubercular activity. In addition, their antifungal and antibacterial activities were evaluated as well. The nicotinohydrazide class displayed different levels of antimicrobial activity and possessed a distinctive pattern of selectivity against the tested microorganisms. However, the 2,6-dichlorobenzylidene counterpart 8b emerged as the most active one in this study, with superior antimycobacterial activity (minimum inhibitory concentration (MIC)=3.90 µg/mL) and potent broad-spectrum antimicrobial activities with MIC range of 0.24–1.95 µg/mL. The structure–activity relationship for such nicotinohydrazides has been established. Further, the cytotoxicity of the most active antitubercular compounds 8b , d and g were tested against the normal breast cells WI-38; none of them displayed significant cytotoxic effect, thereby providing a good therapeutic index.