Encephalitozoon cuniculi can cause serious disease and subclinical infection in rabbits and requires active surveillance to control the infection. This study investigated the association between anti-Encephalitozoon cuniculi antibody status and various health parameters in pet rabbits. A total of 90 rabbits were divided into healthy (N = 30), subclinical (N = 30), and clinical (N = 30) groups based on their anti-Encephalitozoon cuniculi antibody status and clinical presentations. The mean ages of the control (37 ± 40 months) and subclinical groups (38 ± 34 months) were notably lower compared to that of the clinical group (63 ± 38 months, p < 0.01). Serum titers for anti-Encephalitozoon cuniculi antibodies were significantly elevated in rabbits with subclinical and clinical infections compared to those of healthy rabbits (p < 0.05). Neurological signs were predominant in rabbits with active E. cuniculi infection (80.0%), with additional pathological features including urinary dysfunction (10.0%) and cataracts (10.0%). The source of rabbits was not associated with E. cuniculi infection (p = 0.159). Anemia was significantly linked to E. cuniculi infection (p = 0.026); however, no significant associations were found with leukocytosis, thrombocytopenia, or serum biochemistry parameters. Mature adult rabbits were more likely to be infected with E. cuniculi. Recognizing anemia and neurological signs facilitates in early diagnosis of E. cuniculi infection.