The house fly, Musca domestica L., is an important medical and veterinary pest associated with humans and livestock. Management of house flies has relied extensively on chemical control. In this study, we report on the resistance of house fly field-collected populations to diazinon and fenitrothion OP insecticides in Riyadh, Saudi Arabia. The diazinon and fenitrothion median lethal dose (LD50) values against adult female M. domestica field-collected populations were significantly higher than those of the laboratory (LAB) strain. Different levels of resistance were detected in all field-collected populations toward the two OP insecticides. The resistance ratios for diazinon ranged from 62.47 to 309.78, while there were 53.08 to 261.24 for fenitrothion in the eight field-collected populations. The specific activity of acetylcholinesterase (AChE) in all field populations was significantly (p < 0.05) higher than that in the LAB strain. In vitro diazinon and fenitrothion median inhibitory concentration (IC50) values of LAB strain AChE activity were significantly (p < 0.05) lower than those for field-collected populations. This study found high levels of resistance in the house fly field-collected populations to diazinon and fenitrothion. Replacing these two insecticides and any other OPs with novel ones that have different modes of action is an urgent need in the insect-vector control programs in Riyadh, Saudi Arabia. An altered AChE enzyme of M. domestica field populations might be partially responsible for the developed resistance. Monitoring of insecticide resistance development in M. domestica populations and a better understanding of its mechanisms are needed to design operative management strategies for controlling the house flies.