Staphylococcus aureus is an important opportunistic pathogen involved in healthcare and community-associated infections. Mannitol fermentation and coagulase positivity are standard criteria for phenotypic identification, but non-mannitol-fermenting strains can be misidentified. This study aimed to characterize mannitol fermenting and non-mannitol fermenting, coagulase positive Staphylococcus aureus isolates for antimicrobial resistance, biofilm formation and selected resistance- and virulence-associated genes. The total of 262 clinical and non-clinical specimens were examined, isolates were characterized on the basis of growth and fermentation on Mannitol Salt Agar (MSA) and confirmed by tube coagulase test. The susceptibility to 10 different antibiotics was determined and the ability to form biofilm was determined by microtiter plate assay. Six representative isolates (3 mannitol-fermenting and 3 non-mannitol-fermenting) were further subjected to polymerase chain reaction (PCR) to detect the presence of the femA, mecA, icaD, tst-1 and eta genes. Bacterial growth was detected in 212 of 262 specimens. Of 82 Gram-positive isolates that grew on MSA, 55 (67.1%) were coagulase-positive Staphylococcus aureus, including 51 (92.7%) mannitol-fermenting and 4 (7.3%) non-mannitol-fermenting isolates. Resistance rates to cefoxitin and oxacillin were high in both groups (72.5% and 75.0%, respectively), while biofilm production was observed in 84.3% of mannitol-fermenting isolates and 75.0% of non-mannitol-fermenting isolates. Of the 6 isolates analyzed by PCR, all (100%, 6/6) were positive for femA, 66.7% (4/6) were positive for mecA and icaD, 33.3% (2/6) for tst-1, and 16.7% (1/6) for eta. To summarize, despite non-mannitol-fermenting, coagulase-positive Staphylococcus aureus isolates showed significant antimicrobial resistance, biofilm-forming capacity and virulence-associated genes, which highlights the phenotypic diversity of Staphylococcus aureus and emphasizes the importance of combining mannitol fermentation, coagulase testing and molecular characterization to avoid misidentification of atypical isolates.