Consuming food that is rich in antioxidants reduces the risk of developing chronic diseases and oxidative stress. Fruits and vegetables are an excellent source of substances with antioxidant and pro-health properties. Such raw materials, characterized by a high content of polyphenolic compounds and antioxidant capacity, include pear fruits. In this study, the concentrations of bioactive compounds, as well as the antioxidant, anti-inflammatory, and antiproliferative activity in fruits of five selected pear cultivars were determined and compared. LC–MS and UPLC–PDA methods were used to determine the polyphenolic, carotenoid, chlorophyll, and triterpenoid profiles and content, and the antioxidant activity was analyzed using DPPH and ferric-reducing ability of plasma (FRAP) tests. The anti-inflammatory activity was determined against COX-1 and COX-2 enzymes. The cytotoxic activity of the test compounds was assessed against six tumor cell lines. The results showed that the major group of phenolic compounds in all cultivars was phenolic acids. In the group of chromoplastic pigments, chlorophyllide a and 9-cis-β-carotene were the major compounds, while in the triterpene group, ursolic acid was dominant. The antioxidant potential correlated with the content of polyphenols and carotenoids, and was the strongest for the ‘Radana’ cultivar. The highest antiproliferative activity in all varieties was established for bladder cancer.