Methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening infections. Zinc oxide is well known as an effective antibacterial drug against many bacterial strains. We investigated the performance of zinc oxide nanorods synthesized by Albmiun as a biotemplate as an antibacterial drug in this study; the fabrication of zinc oxide nanorods was synthesized by sol-gel methods. We performed physicochemical characterization of zinc oxide nanorods by physiochemical techniques such as FTIR spectroscopy, X-ray diffraction, and TEM and investigation of their antimicrobial toxicity efficiency by MIC, ATPase activity assay, anti-biofilm activity, and kill time assays, as well as the mecA, mecR1, blaR1, blaZ, and biofilm genes (ica A, ica D, and fnb A) by using a quantitative RT-PCR assay and the penicillin-binding protein 2a (PBP2a) level of MRSA by using a Western blot. The data confirmed the fabrication of rod-shaped zinc oxide nanorods with a diameter in the range of 50 nm, which emphasized the formation of zinc oxide nanoparticles with regular shapes. The results show that zinc oxide nanorods inhibited methicillin-resistant S. aureus effectively. The MIC value was 23 μg/mL. The time kill of ZnO-NRs against MRSA was achieved after 2 h of incubation at 4MIC (92 μg/mL) and after 3 h of incubation at 2MIC (46 μg/mL), respectively. The lowest concentration of zinc oxide nanorods with over 75% biofilm killing in all strains tested was 32 μg/mL. Also, we examined the influence of the zinc oxide nanorods on MRSA by analyzing mecA, mecR1, blaR1, and blaZ by using a quantitative RT-PCR assay. The data obtained revealed that the presence of 2× MIC (46 μg/mL) of ZnO-NRs reduced the transcriptional levels of blaZ, blaR1, mecA, and mecR1 by 3.4-fold, 3.6-fold, 4-fold, and 3.8-fold, respectively. Furthermore, the gene expression of biofilm encoding genes (ica A, ica B, ica D, and fnb A) was tested using quantitative real-time reverse transcriptase-polymerase chain reaction (rt-PCR). The results showed that the presence of 2× MIC (46 μg/mL) of ZnO-NRs reduced the transcriptional levels of ica A, ica B, ica D, and fnb A. Also, the PBP2a level was markedly reduced after treatment with ZnO-NRs.