Background: Fusidic acid-resistant Staphylococcus aureus (FRSA) has been reported in many countries to have a remarkable difference in resistance determinants. Fusidic acid resistance is very important because it might lead to the failure of topical treatment, especially when it is used as empiric therapy. In addition, its resistance might be linked to other antibiotic resistances. The overall rate of fusidic acid resistance is still relatively low. However, there is an increase in the prevalence of clinical isolates of FRSA worldwide. Objectives: We aimed to characterize FRSA isolated from Jordanian patients and evaluate the occurrence of the genetic resistance caused by fusB and fusC. Methods: We conducted a prospective cross-sectional study to determine the prevalence and the resistance pattern of S. aureus to fusidic acid among Jordanian patients and healthy people. Staphylococcus aureus clinical isolates (n = 113) obtained from patients admitted to Prince Hamzah Hospital between February and July 2015 were compared with isolates (n = 288) obtained from healthy subjects. Conventional methods were used for the identification of S. aureus and further confirmations were done by the existence of the thermonuclease gene using polymerase chain reaction (PCR). Screenings of antibiotic resistance were performed using the disc diffusion method. The minimum inhibitory concentrations were calculated using the E-test. PCR was used to detect the presence of resistant genes. Results: The FRSA frequency was significantly higher among clinical isolates (31.9%) than among isolates from healthy subjects (1%) and in methicillin-resistant Staphylococcus aureus (MRSA) (66.7%) than in methicillin-sensitive Staphylococcus aureus (MSSA) (33.3%). Of the FRSA isolates, 38.9% and 16.7% carried fusB and fusC, respectively, and they displayed low resistance compared to non-fusB, non-fusC FRSA isolates. The rate of FRSA was significantly (P < 0.05) higher among MRSA than among MSSA isolates (n = 24, 66.7% and n = 12, 33.3%, respectively). We found no association between fusidic acid determinants among MRSA and MSSA (P > 0.05). Conclusions: A high occurrence of FRSA was detected in Jordanian clinical isolates of S. aureus, particularly among MRSA. Moreover, fusB was the predominant resistance determinant, with low-level resistance. Based on our findings, fusidic acid susceptibility testing is strongly recommended in medical laboratories. The restricted use of fusidic acid is advised.