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<i>Tropheryma whipplei</i> (TW), <i>Helicobacter pylori</i> (HP), and intestinal protozoa (IP) are widespread pathogens with similar routes of transmission and epidemiological risk factors. Epidemiological data on co-infection between TW, HP, and IP are scarce. We aim to more deeply investigate the co-infection rate for these pathogens, evaluating the risk factors and symptoms. Methods: This is a cross-sectional study conducted at the IRCCS Sacro Cuore Don Calabria Hospital in Northern Italy, a referral center for tropical and Whipple’s disease (WD). Stored stool samples from 143 subjects previously tested for TW DNA by real-time PCR were explored for HP and IP DNA detection. The virulence factor <i>cagA</i> was investigated in HP-positive patients. Results: A history of migration was reported significantly more in TW-positive than in negative subjects (34.1% vs. 9.1%, <i>p</i> = 0.001) and in HP-infected than in those non-infected (59.1% vs. 9.1%, <i>p</i> < 0.001). The HP infection rate differed significantly between TW-infected and uninfected groups (31.8% vs. 8.1%, <i>p</i> = 0.001), while no difference was observed for IP infection. Significantly higher TW intestinal colonization was found in HP-infected patients than in non-infected (63.6% vs. 24.8%, <i>p</i> < 0.001). In addition, the proportion of Blastocysts positive finding was also significantly higher in HP-infected than in non-infected (40.9% vs. 17.4%, <i>p</i> = 0.018). Conclusions: The present study is the first to report a high TW and HP co-infection rate. To reduce the risk of morbidity from a chronic infection of either pathogen, clinicians may consider TW-HP molecular screening on the same stool sample for patients with suspected HP disease or WD, particularly in case of travel history.