The tumor suppressor gene TP53 and its downstream genes P21 and MDM2 play crucial roles in combating DNA damage at the G1/S cell cycle checkpoint. Polymorphisms in these genes can lead to the development of various diseases. This study was conducted to examine a potential association between tobacco substance usage (TSU) and single-nucleotide polymorphism (SNP) at the exon regions of the P53, P21, and MDM2 genes by comparing populations of smokers and non-smokers from Saudi Arabia. P53 rs1042522 (C/G), P21 rs1801270 (A/C), and MDM2 rs769412 (A/G) were investigated by genotyping 568 blood specimens: 283 from male/female smokers and 285 from male/female non-smokers. The results obtained from the smokers and their control non-smokers were compared according to age, sex, duration of smoking, and type of TSU. Heterozygous CG, homozygous GG, and CG+GG genotypes, as well as the G allele of rs1042522 were significantly associated with TSU in Saudi smokers compared with non-smokers. The C allele frequency of rs1801270 was also associated with TSU in smokers (OR = 1.33, p = 0.049) in comparison with non-smokers, in younger smokers (≤29 years) (OR = 1.556, p = 0.03280) in comparison with non-smokers of the same age, in smokers who had smoked cigarettes for seven years or less (OR = 1.596, p = 0.00882), and in smokers who had consumed shisha (OR = 1.608, p = 0.04104) in comparison with the controls. However, the genotypic and allelic frequencies for rs769412 did not show significant associations with TSU in Saudis. The selected SNP of P53 was strongly associated with TSU and may be linked to TSU-induced diseases in the Saudi Arabian population.