The presence of inhibitory substances in milk arises in consumers public health hazard. Some certain chemical substances are used as preservatives to extend the shelf life of milk. In the present study, 160 milk samples were collected from street vendors, farmer's houses, shop keepers and dairy farms (40 samples of each). All investigated milk samples were subjected to inhibitory substance detection using Wynther Blyth test and the qualitative B. subtilis disc assay method. Chemically, the present samples were subjected to Hehner's test for detection of formalin, modified Piens test for hydrogen peroxide, glycerin test for boric acid and salicylic acid test. All examined milk samples were free from salicylic acid. Dairy farm milk samples proved to be free from chemical preservatives while, street vendors milk was the worst. 35% of their samples contained inhibitory substances using the Wynther Blyth test. Moreover, 10, 15 and 2.5% of these samples contained formalin, boric acid and hydrogen peroxide, respectively. Qualitative B. subtilis disc assay method concluded that 30, 10, 15 and 5% of street vendors, farmer's houses, shop keepers and farm milk samples contained inhibitory substances, respectively. Correlation between the results of specific tests for chemical preservatives and that obtained by the Wynther Blyth test and the disc assay method indicated that chemical preservatives could be detected by both tests in 90.9, 25, 66.6% of street vendors, farmer's houses, shop keepers milk samples, respectively. Moreover, the Wynther Blyth test proved to be more sensitive for detection of chemical preservatives than the qualitative B. subtilis disc assay method. It was concluded that these inhibitory substances are very objectionable and illegal substances, so they should be screened randomly and periodically. Moreover, intensification of the education among street vendors and shop keepers would greatly reduce the occurrence of these substances in milk.