This investigation was aimed at studying resistance marker loss of multidrug resistant (MDR) Staphylococcus aureus strains after treatment with sub-clinical dilutions of acridine orange. Seven (7) pure axenic strains of Staph aureus coded SA1-SA7 isolated from seven infected human sources which included seminal fluid, ear swab, midstream urine, wound swab, urethral swab, high vaginal swab and endocervical swab respectively, were obtained from the Medical Microbiology laboratory of the Delta state University Teaching Hospital, Oghara, Nigeria and stocked on sterile Nutrient agar slants. Colonies from resulting stocked cultures were re-confirmed by subculturing them on sterile Mannitol Salt agar plates and incubated at 37oC for 24hours. Gram staining, catalase and coagulase tests were carried out on resulting colonies. Antibiotic sensitivity test by agar disc diffusion method was done on confirmed strains on sterile Mueller Hinton agar plates before and after treatment with acridine orange (AO). Staph aureus strains that showed ≤50.0% reduction in resistance marker after treatment with 0.35, 0.55, 0.75 and 0.95ug/ml dilutions of AO were noted. Minimum inhibitory concentration (MIC) assay was done using gentamicin on SA1 strain with all four dilutions of AO. All seven strains showed resistance against augmentin, amoxicillin and cloxacillin. The highest (19.9± 2.3mm) and lowest (6.6 ± 7.3mm) zones of inhibition recorded by all the strains were reactions to chloramphenicol and cotrimoxazole respectively. Strains SA4, SA2 and SA7 recorded 17.0±4.9mm, 14.4±3.3mm, 14.0±5.9mm and 7.3±4.6mm mean zones of inhibition to gentamicin, erythromycin, chloramphenicol and tetracycline respectively. Strains SA2, SA4 and SA7 were each resistant to three of the total antibiotics used. Strains SA6, SA1, SA3 and SA5 were resistant to four, five, five and six of the antibiotics used respectively. Mean loss of 50.0% or more of resistance marker (RM) was recorded after treatment with 0.35ug/ml (60.0%), 0.95ug/ml (52.7%) and 0.75ug/ml (52.2%) to erythromycin. There was zero loss of RM to gentamicin, chloramphenicol and tetracycline after treatment with 0.55ug/ml, 0.35ug/ml and 0.75ug/ml AO respectively. Acridine orange dilutions of 0.95 and 0.35ug/ml produced an eight-fold (1.25ug) and four-fold (2.5ug) reduction respectively in the minimum inhibitory concentration of gentamicin. The implications of these findings are discussed.