In exploring the biofouling resistance capabilities of biomimetic superhydrophobic surfaces, femtosecond laser technology was employed to create these surfaces, leading to comprehensive anti-fouling efficacy evaluations. Laboratory tests assessing surface contact angle, roughness, and chemical stability were conducted, alongside extended testing in simulated marine and freshwater environments. These tests aimed to determine whether these surfaces could maintain their anti-fouling properties under various environmental conditions. The results from controlled laboratory conditions indicated that these surfaces exhibited excellent hydrophobicity and chemical stability, suggesting potential effectiveness against biofouling. However, when subjected to more complex, real-world aquatic settings, the performance of these surfaces was not as effective as anticipated. Initially, the surfaces showed promise in resisting fouling, but over time, their effectiveness significantly diminished. This decline in performance was attributed to the accumulation of biofouling agents, such as proteins and polysaccharides, which facilitated the adhesion of various fouling organisms. This gradual buildup of biological material highlighted a critical limitation of superhydrophobic surfaces in dynamic aquatic environments. These findings challenge the previously held assumption that superhydrophobic properties alone are sufficient for effective biofouling resistance. It becomes evident that environmental factors play a significant role in the performance of these surfaces. This study underscores the need for future research to focus on the environmental impact on anti-fouling surfaces and to explore the integration of superhydrophobic features with other anti-fouling technologies. Such multidisciplinary approaches could lead to the development of more effective and durable solutions to combat biofouling, which is a persistent problem in marine and freshwater systems.