There is a new approach to prevent the proliferation of food microorganisms using of plant essential oils. Commiphora myrrha is considered as highly effective, natura, and safe antimicrobial. Therefore, the current study aimed to evaluate the effects of Commiphora myrrha on (1) pathogenic positive bacteria including Listeria monocytogenes, Staphylococus aureus, and Bacillus cereus as well as gram-negative bacteria including Escherichia coli O157:H7 and Salmonella typhimurium, and (2) the sensory properties of processed cheese spreads at 5 different levels of usage. Moreover, processed cheese spread (PCS) samples were prepared by using 5 different ratios of Commiphora myrrha essential oil at 0.2, 0.4, 0.6, 0.8, 1 w/w to evaluate its organoleptic acceptability. The PCSs was made by heating and mixing the ingredients at 85°C for 10 min. Gross chemical composition and some physical characteristics were estimated through 3 mo of storage at 5 ± 1°C. Oil separation, penetration, melting point and color parameters were also determined through the storage period. Results showed that all tested microbes were susceptible to Commiphora myrrha at minimum inhibitory concentration of 2–5 µL of oil/mL. Moreover, results showed that using 0.2% (w/w) Commiphora myrrha essential oil gave satisfactory sensory properties. The appearance was shiny; gumminess and oil separation were absent. The overall preference of preferable ratio was “like very much” comparing to the other treatments. Color was more acceptable without significant differences (P > 0.05) and it spread well compared with control. The penetration values were 33.5, 32.0 and 31.2 mm versus 33.0, 30.5 and 26.5 mm for treated PCS versus control PCS (P > 0.05) when fresh, and after 1 mo and 3 mo of storage. Moreover, meltability 85.4, 81.6 and 80.0 mm versus 81.6, 80.5 and 78.7 mm for treated PCSs compared with control when fresh, and after 1 moand 3 mo of storage. Commiphora myrrha essential oil can be used successfully as antimicrobial agent during preparing of PCS with acceptable properties and satisfied sensory behavior.