Cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-( N-methyl)isatin (HL 1 and HL 2, respectively) were synthesized and characterized, being the crystal structures of HL 1, HL 2 and [Ni(L 1) 2] · 2CHCl 3 elucidated by X-ray diffraction techniques. The in vitro antimicrobial activity of all these compounds was tested against several bacteria and fungi. HL 1and its complexes exhibited a strong inhibition of the growth of Haemophilus influenzae (MIC 0.15–1.50 μg/mL) and good antibacterial properties towards Bacillus subtilis (MIC 3–25 μg/mL). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound inhibiting the growth of each strain. The antibacterial effectiveness was confirmed against a number of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Yeasts and moulds showed a low susceptibility, except the dermatophyte mould Epidermophyton floccosum that is inhibited at concentrations ranging from 6 to 50 μg/mL. In general, the antimicrobial activity of the thiophene derivatives was greater than that of the isonicotinic analogues.