Background: In the past vicennium, several artificial intelligence (AI) and machine learning (ML) models have been developed to assist in medical diagnosis, decision making, and design of treatment protocols. The number of active pathologists in Poland is low, prolonging tumor patients’ diagnosis and treatment journey. Hence, applying AI and ML may aid in this process. Therefore, our study aims to investigate the knowledge of using AI and ML methods in the clinical field in pathologists in Poland. To our knowledge, no similar study has been conducted. Methods: We conducted a cross-sectional study targeting pathologists in Poland from June to July 2022. The questionnaire included self-reported information on AI or ML knowledge, experience, specialization, personal thoughts, and level of agreement with different aspects of AI and ML in medical diagnosis. Data were analyzed using IBM®SPSS®Statistics v.26, PQStat Software v.1.8.2.238, and RStudio Build 351. Results: Overall, 68 pathologists in Poland participated in our study. Their average age and years of experience were 38.92 ± 8.88 and 12.78 ± 9.48 years, respectively. Approximately 42% used AI or ML methods, which showed a significant difference in the knowledge gap between those who never used it (OR = 17.9, 95% CI = 3.57–89.79,p< 0.001). Additionally, users of AI had higher odds of reporting satisfaction with the speed of AI in the medical diagnosis process (OR = 4.66, 95% CI = 1.05–20.78,p= 0.043). Finally, significant differences (p= 0.003) were observed in determining the liability for legal issues used by AI and ML methods. Conclusion: Most pathologists in this study did not use AI or ML models, highlighting the importance of increasing awareness and educational programs regarding applying AI and ML in medical diagnosis.