Biomonitoring of people exposed to hazardous materials provides opportunities for early identification of several diseases, particularly in those individuals who are constantly exposed to pesticides, such as pesticide operators and workers in pesticide manufacturing industry. However, data on this hot topic are limited in Pakistan. In this study, insecticide toxic effects and biochemical alterations (i.e., damage in DNA and enzyme activity) were studied in blood samples of occupationally exposed individuals from Punjab, Pakistan. Eight out of twenty-seven blood samples (29.6%) of the pesticide operators were found positive in five insecticides, with the maximum concentration found for chlorpyrifos-methyl (0.039 μg/mL). Eleven out of twenty-seven blood samples (40.7%) of the pesticide industry workers were found positive in eight insecticides, with the maximum concentration found for endosulfan (0.051 μg/mL). Comet tail length was 16.88 ± 4.57 μm in pesticide industry workers and 16.33 ± 3.78 μm in pesticide operators, which were significantly higher (P < 0.01) than that recorded in the control group (4.84 ± 2.21 μm). Values of serum cholinesterase (SChE) concentration were slightly lower (P > 0.05) in exposed individuals, whereas values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) concentration were significantly higher (P < 0.01) in exposed individuals compared with control group. Exposure duration and total insecticide concentration in blood samples were positively associated with comet tail length, ALT activity, AST activity, and ALP activity, but negatively with SChE. DNA damage was higher in smokers vs. non-smokers. Also, a positive association was found between comet tail length and number of cigarettes per day. Overall, occupational exposure to insecticides can pose serious health risks to pesticide operators and workers in pesticide manufacturing industry, highlighting the necessity of personal protection in those groups for preventing exposure and resultant health disorders.