In the present investigation, fourteen new alkylthio substituted 5,6-dimethyl-1,4-benzoquinone derivatives were synthesized as potential antimicrobial and antifungal agents. First, 2,3-dichloro-5,6-dimethyl-1,4-benzoquinone was reacted with alkyl thiols containing ester group to yield its mono-and bis(thio) derivatives and then these compounds were investigated for their antibacterial and antifungal activities. In vitro studies were carried out using four Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 4352, Proteus mirabilis ATCC 14153), three Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 29213), and two fungi (Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019) by using the microbroth dilutions technique. Most of the synthesized compounds (3a-c, 3f, 4b-c, and 4e) exhibited good antibacterial activities with MIC values between 9.76 and 78.12 mu g/mL. Compound 4e was identified as a hit compound with antibacterial efficiency against P. aeruginosa and E. faecalis with MIC value of 9.76 and 19.53 mu g/mL, respectively. (C) 2019 Elsevier B.V. All rights reserved.