This study aimed to provide information on the presence and frequency of viral and parasitic agents in wildlife presented to a Veterinary Teaching Hospital in 2020-2021. Serum and faecal samples were collected from 50 rescued animals (roe deer, fallow deer, foxes, badgers, pine martens, and porcupines) and examined by serological, molecular, and parasitological techniques. Transtracheal wash (TTW) was also collected post-mortem from roe deer. Overall, the results of the different techniques showed infections with the following viral and parasitic agents: Bovine Viral Diarrhea Virus, Small Ruminant Lentiviruses, Kobuvirus, Astrovirus, Canine Adenovirus 1, Bopivirus, gastrointestinal strongyles, Capillaria, Ancylostomatidae, Toxocara canis, Trichuris vulpis, Hymenolepis, Strongyloides, Eimeria, Isospora, Dictyocaulus, Angiostrongylus vasorum, Crenosoma, Dirofilaria immitis, Neospora caninum, Giardia duodenalis, and Cryptosporidium. Sequencing (Tpi locus) identified G. duodenalis sub-assemblages AI and BIV in one roe deer and one porcupine, respectively. Adult lungworms collected from the TTW were identified as Dictyocaulus capreolus (COX1 gene). This is the first molecular identification of G. duodenalis sub-assemblage AI and D. capreolus in roe deer in Italy. These results show a wide presence of pathogens in wild populations and provide an overview of environmental health surveillance.