Ocular toxoplasmosis (OT) is caused by protozoan T. gondii. Ophthalmological examination is considered the gold standard for OT diagnosis, and laboratory tests are used for diagnostic confirmation. However, these tests can present different results, which change depending on their basis, on sample type and on patients’ clinical alteration. Thus, the aim of the present study is to assess immunodiagnostic and molecular techniques applied in blood, serum and tear fluid to diagnose T. gondii infection in patients seen at an Ophthalmology Clinic. In total, 160 patients were included in the study, 40 of them had OT with active lesions (G1); 40 had OT with healed lesions (G2), 40 had non-toxoplasmic uveitis (G3) and 40 had no ocular alterations (G4). Serum samples were subjected to Immunoenzymatic Assay (ELISA) and to Indirect Immunofluorescence Reaction (IFAT) to search for anti-T. gondii IgM and IgG. Tear fluid samples were analyzed through ELISA for IgA research. All blood and tear fluid samples were subjected to conventional polymerase chain reaction (cPCR) and in a Nested PCR model for T. gondii DNA amplification with targets B1, GRA7 and REP 529. IgG and IgM anti-T. gondii was detected in serum samples from 106 and 15 patients, respectively, when combining ELISA and IFAT results. Anti-T.gondii IgA antibodies were detected in 9.2% of the tear material. Nested PCR with GRA7 target showed higher positivity in blood samples (24.4%); Nested PCR with B1 target showed a higher frequency of positivity in tears (15%). Biological samples of patients with active lesions showed the highest positivity frequencies in all immunodiagnostic assays, as well as in most PCR models. The present results highlighted the need of associating techniques with different fundamentals to confirm OT diagnosis. Furthermore, further tear fluid analyses should be performed to validate this biological material as lesser invasive alternative for the more accurate OT diagnosis.