Prostatic hyperplasia (PH) is the most common reproductive disorder in dogs and can lead to discomforting problems such as haematuria, urinary incontinence, constipation, difficulty in defecating and stiffness of the hind limbs. The diagnosis of PH is nowadays based on digital rectal examination (DRE), ultrasonography (US) and radiography (X-ray). However, markers associated with PH are barely used for diagnostic purposes. Recently, there have been reports on the use of certain biomarkers for diagnosing PH in dogs such as canine PSA (Prostate Specific Antigen), microRNA and vascular endothelial growth factor (VEGF). Nevertheless, it has been generally accepted that these biomarkers play only an auxiliary role. Accordingly, the aim of our study was to evaluate the usefulness of the CCL11 (eotaxin-1) and TGF-beta 1 markers, which are used in the diagnosis of prostate diseases in humans, in case of dogs with PH. The study was carried out on 40 dogs of different breeds divided into three groups. Group I (n = 9) comprised dogs up to 5 years of age without changes indicative of PH. Group II (n = 17) included dogs aged 5–10 that were examined and diagnosed with (PH) and Group III (n = 14) which consisted of dogs over 10 years of age who were also diagnosed with PH. The study demonstrated that CCL11 levels did not differ significantly between the study groups and the median levels were 7.27 pg/mL, 7.57 pg/mL, 6.81 pg/mL, and IQR ranges 1.55 pg/mL, 1.74 pg/mL, 2.32 pg/mL, respectively. In contrast, TGF-beta 1 levels were detectable only in 6 dogs of group III and averaged the median of 28.86 pg/mL, IQR ranges 10.07 pg/mL. The study proved that CCL11 and TGF-beta 1 markers are of a limited use when diagnosing PH in dogs as no significant correlation related to age, body weight or prostate size was found.