This study presents the synthesis and spectral analysis of new derivatives of 1,2,4-triazole-3-thione and 1,3,4-thiadiazole. New compounds were prepared by cyclization reaction of acyl thiosemicarbazide derivatives in the presence of alkaline and acidic media. All synthesized compounds were screened for their in vitro antibacterial activity by using the agar dilution technique. Six of the compounds had potential activity against Gram-positive bacteria (minimal inhibitory concentration [MIC] = 15.63–500 μg/mL). Some compounds showed good activity especially against Bacillus subtilis ATCC 6633 (MIC = 15.63–250 μg/mL), Staphylococcus aureus ATCC 25923 (MIC = 31.25–250 μg/mL), and Micrococcus luteus ATCC 10240 (MIC = 125–250 μg/mL). Graphical abstract