Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.