Background: Encephalitozoon cuniculi can affect a number of animals. Currently, there is limited clinical information on E. cuniculi in guinea pigs. Case description: A 3-year-old guinea pig with torticollis, dehydration, and hypothermia was brought to the clinic. After symptomatic treatment, the animal's status improved, and the animal returned home. Two computed tomography scans over a period of 23 days revealed oval hypodense lesions in the right temporal lobe, which enlarged over time. The animal was euthanized. A histopathological examination revealed focal granulomatous encephalitis, and PCR allowed the detection of E. cuniculi DNA in the brain tissue. It was found that the possible cause of the disease was E. cuniculi invasion. Conclusions: Based on this report, E. cuniculi should be considered a differential in guinea pigs with granulomatous encephalitis.