High-grade serous ovarian carcinoma (HGSOC) remains a major challenge in oncology. This study aimed to assess the spatial and phenotypic heterogeneity of EpCAM, HER2, TROP2, and pan-cytokeratin expression in primary tumor cells, peritoneal metastases, and the ascitic tumor compartment (ascites), comprising spheroids and single cells, and to determine whether the identified subpopulations were associated with neoadjuvant chemotherapy (NACT) and disease progression. The study is prospective, single-center; it involved 27 patients with histologically confirmed HGSOC; EpCAM, HER2, TROP2, and pan-cytokeratin expression were assessed by immunofluorescence. The proportions of EpCAM−HER2− cells were different in the primary tumor, peritoneal metastases, and ascites, and were highest in single cells from ascitic fluid and metastatic tissue (p = 0.0012). CK–TROP2– cells were more frequent among single cells (73.50% [51.00–91.25%]; p = 0.0249). The proportion of CK+TROP2– cells was higher in solid tumor lesions, accounting for 83.00% [65.00–95.00%] of cells in primary tumors and 94.50% [47.00–98.00%] in metastatic tissue (p < 0.0001). In patients who received NACT, the proportion of EpCAM+HER2+ cells in primary tumors and metastatic lesions was higher than in patients who did not undergo pre-surgery therapy (42.50% [6.25–71.25%] vs. 0.00% [0.00–5.00%]; p = 0.0333). The clinical significance of the EpCAM−HER2− subpopulation depended on its organization within ascites. An increased proportion of EpCAM−HER2− cells among single ascitic cells, but not within spheroids, was independently associated with a higher likelihood of progression (OR [95% CI], 1.256 [1.010–7.769]; p = 0.0322) and shorter progression-free survival (OR [95% CI], 1.118 [1.001–1.518]; p = 0.0459). HGSOC is characterized by pronounced phenotypic heterogeneity across tumor compartments. The ascites may reflect the phenotypic state of disseminated tumor cells.