Antimicrobial activity in vitro of a series of novel azaphenothiazine derivatives containing a quinoline moiety was investigated using Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) strains as well as in Candida albicans yeast. The examined compounds showed the highest activity against Enterococcus faecalis and Escherichia coli whereas activity against Pseudomonas aeruginosa was the lowest. Compound 1d demonstrates the highest activity against all tested bacterial strains. Compounds 1c, 1h and 1k with various substituents (CH3, OH, NH2) at C11 position of the quinobenzothiazine ring, did not exhibit activity against any tested bacterial strain. Only compounds 1m and 1n with long aliphatic chains at the quinoline nitrogen atom showed antifungal activity. Correlations between antimicrobial activity and chemical structure of the tested compounds were observed.