Conjunctival intraepithelial lymphocytes, tear soluble molecules and commensal microbiota have important roles in the ocular mucosal immune response in healthy and diseased subjects. For the purpose of this study, the cellular and microbial populations of the conjunctiva and the lacrimal soluble molecules were analyzed to find the main biomarkers in allergic conjunctivitis. A total of 35 healthy subjects, 28 subjects with seasonal allergic conjunctivitis and 32 subjects with perennial allergic conjunctivitis were recruited to obtain peripheral blood, conjunctival brush cytology, tear fluid and microbiota samples. Flow cytometry for lymphocytes, multiplex bead assays for cytokines and high-throughput DNA sequencing for microbiome analysis were used. For perennial allergic conjunctivitis, an increased proportion of Th2 and NKT lymphocytes was found, while CD3+TCRγδ+ lymphocytes and double negative MAIT cells were decreased. In contrast, seasonal allergic conjunctivitis was distinguished by an increase in Th17 and Th22 cell proportions, while the Th1 cell proportion decreased. Among tear fluid, the vast majority of pro-inflammatory cytokines (especially Th2 and Th17 cytokines) in perennial allergies and MMP-9 together with IgA in seasonal allergies were increased. In contrast, TGF-β2 was decreased in both forms of conjunctivitis. Finally, fungal (Malassezia species) and bacterial (Kocuria and Propionobacterium acnes species) colonization were observed in the perennial allergic conjunctivitis group. These results provide the basis for the development of a disease profile for perennial allergic conjunctivitis and open the door to new therapeutic and diagnostic strategies.