Cosmetic products, such as mascaras and eyeliners, can become contaminated with pathogenic microorganisms through repeated use by different individuals. This study aimed to investigate the microbial contamination of in-use mascara and eyeliner products and its correlation to the types of preservatives used. Various brands of waterproof and non-waterproof mascaras and eyeliners were obtained from local markets in Jordan. Swab samples were collected before distribution to female volunteers, who used the products as they normally would. Additional samples were taken after one, two, and three months of use, resulting in a total of 96 samples. Quantitative evaluation of bacterial growth was performed by culturing the swabs, while qualitative analysis identified isolates to the species level. The results showed waterproof products had higher bacterial counts that increased over time. The most prevalent contaminants were gram-positive bacilli and cocci, including Bacillus cereus. (B.cereus), Brevibacillus brevis (B.brevis), Staphylococcus aureus (S.aureus) and Staphylococcus epidermidis (S.epidermidis), which are potential pathogens. Preservative efficacy was assessed through challenge test. The Phenoxyethanol-Potassium Sorbate combination was most effective in waterproof mascara, keeping bacterial numbers low (8x102) with no uncountable growth until after three months. Non-waterproof mascara benefited from Phenoxyethanol-Stearic Acid, exhibiting no growth for three months. In waterproof eyeliner, Potassium Sorbate and Phenoxyethanol prevented growth, while non-waterproof eyeliner remained bacteria-free with Propylene Glycol, Isopropyl paraben, Butyl Paraben, and Iso Butyl Paraben. This study highlights the importance of maintaining proper hygiene practices and implementing strict quality control measures during the production and use of cosmetics. The preservative efficacy challenge test emphasizes the need for appropriate preservatives to prevent microbial contamination and growth in these products