In the textile and fashion industry, cotton knitted fabric is commonly used for making apparel. To achieve different functional effects of the cotton knitted fabric, chemical treatments are commonly used, but due to increased environmental restrictions, physical treatment, e.g., with a laser, has recently been introduced to the market as an environmentally friendly way to treat the surface of cotton fabric. Thus, the aim of the present study is to evaluate the impact of laser treatment on the physical and mechanical properties of 100% cotton single jersey knitted fabrics with different yarn counts. The fabrics had been treated using a carbon dioxide laser (CO2) with different levels of intensity in terms of the resolution and treatment time, i.e., the resolution (28, 32, 36, 40 dpi) and pixel time (100, 110, 120, 130 μs). The laser power density resulting from the different combinations of laser variables was measured. Various evaluation tests including scanning electron microscopy, fabric weight, fabric thickness and fabric bursting strength were used to study the effect of laser treatment on the knitted fabrics. Grooves and microcracks appeared on the fibers after laser treatment. With increasing resolution and pixel time, these changes became more distinct on the fiber surface. The highest number of pores was found on the yarn with a higher yarn count and lower yarn twist. According to the results of knitted fabrics treated with a laser, the largest difference was found for the aspects of weight, thickness and whiteness values of the fabric knitted with the thickest yarn. Furthermore, it was confirmed that not only the yarn count affects the change in weight and thickness, but also the fabric structure such as the number of courses affected the laser treatment result.