The increasing resistance of pathogenic bacteria and fungi to remedies and their biofilm formation made it is obligatory to search for new, safer and more effective alternatives using medicinal plants. The present investigation aims to study the potency of Syzygium aromaticum (clove) aqueous extract mixture with antibiotics or rotaxane derivatives against 3 multi-drug resistant bacteria, 2 fluconazole resistant fungi strains, their biofilm, 3 aflatoxigenic Aspergillus flavus strains and aflatoxin biosynthesis. Some bioactive compounds and elements were obtained for clove extract by Fourier transform infrared (FTIR) spectroscopy and energy dispersive X-ray fluorescence (EDXRF) analysis and gas chromatography-mass spectroscopy (GC-MS). A 33.3 - 60 mg mL−1 concentrations of S. aromaticum aqueous extract were sufficient as MBC against E. coli O157:H7, E. coli O121, and Staphylococcus aureus (MRSA) strains, respectively. In practically all combinations of extract, antibiotics, and rotaxane derivatives, synergistic or indifferent relations were detected. No antagonistic interactions were observed against bacteria. For C. albicans and C. glabrata concentrations 24.64 and 49.28 mg mL−1 of clove were observed as MFC, respectively. Conversely, A. flavus strains were not repressed. Combination of clove with fluconazole exposed synergistic interactions against C. albicans. Clove considerably suppressed biofilm formation with inhibition percentage of 81.99, 82.2, and 72.89% for E. coli (O157:H7 and O121) and S. aureus (MRSA), respectively. Furthermore, C. albicans biofilm was reduced and the inhibition percentage was 54%. The highest inhibition percentage of aflatoxin production using clove was 31.7%. This investigation suggests the clove aqueous extract to be a prospective broad spectrum antimicrobial composite alone or in combination.