The composition of the ovarian fluid (OF) is predestined for egg storage and for prolonging the fertilization period during natural spawning as well as during artificial fertilization. A thorough characterization of the specific components within the OF and eggs may provide deeper knowledge about their functions, a better understanding of egg-OF signaling, and the origin of the OF proteins, as well as reveal potential biomarkers of oocyte quality and aging. To uncover the protein composition of pikeperch OF and eggs, we performed an in-depth comparative quantitative label-free proteomics analysis using liquid chromatography mass spectrometry of OF and egg samples collected from the same pikeperch females. A total of 796 ovarian fluid and 1296 egg proteins were identified. The 50 most abundant proteins with prevalent protective roles were common to pikeperch OF and eggs, which may suggest that eggs are safeguarded internally and externally and can indicate functional interconnection between those two important reproductive components. A total of 624 differentially abundant proteins between OF and eggs were also identified. The proteins that were more abundant in the OF are known to actively participate in egg immunological defense, scavenge free radicals and play an important role in the restoration of immune homeostasis after inflammation and in modulating sperm performance during pikeperch fertilization. In turn, several proteins more abundant in eggs are members of signaling pathways related to extensive synthesis of maternal RNAs, proteins, and lipids that are used in oocyte maturation and fertilization, to secure antioxidant machinery against oxidative stress to maintain gamete quality and with a potential role in the regulation of meiotic cell cycle progression during oocyte maturation and its resumption following fertilization. Our results suggest that the identified OF proteins are derived from eggs, the epithelium of the ovarian cavity, blood and possibly extracellular vesicles. This OF and egg protein dataset is the largest described to date in teleost fish, which significantly extends the known protein components of female fish reproduction. Moreover, the obtained results provide a useful groundwork for future experimental approaches aimed at identifying proteins that enhance sperm motility parameters during fertilization and unraveling the content of external vesicles and their role in growth and oocyte maturation in fish. Data are available via ProteomeXchange with identifier PXD026245.