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Potentiated sulfonamides, such as sulfadiazine-trimethoprim (SDZ-TRIM), are frequently used antimicrobials in both human and veterinary medicine. To optimise their use in relation to the emerging problem of resistance selection, this paper studied the impact of dose and administration route of SDZ-TRIM on the exposure of the gut microbiota to these antimicrobials. An animal experiment was conducted with 36 pigs, divided into six different treatment groups (n = 6). Three different administration routes were outlined: oral (PO) gavage, intramuscular (IM) injection and medicated feed, with 5-day therapy duration. Conventional dosing (30 mg SDZ-TRIM/kg bodyweight [BW]) and half dosing (15 mg SDZ–TRIM/kg BW) was performed for the oral routes in two applications per day. For the IM route, a conventional dose of 15 mg SDZ-TRIM/kg BW or a double dose of 30 mg SDZ–TRIM/kg BW was administered once daily. After daily collection of blood and faeces, the intestinal content of all animals was sampled in different gastrointestinal tract (GIT) segments, and SDZ and TRIM were quantified. Remarkably, SDZ accumulated in distal GIT segments, independently of the administration route. High concentrations (mean ± standard deviation) up to 26.93 ± 8.36 µg/g, 11.15 ± 3.78 µg/g and 19.36 ± 1.86 µg/g after PO gavage, IM administration and medicated feed, respectively, were measured for SDZ. In contrast, TRIM concentrations decreased from proximal to distal segments and were mostly below the limit of quantification (0.025 µg/g). The high oral bioavailability of SDZ indicates gastrointestinal secretion is a substantial elimination route for SDZ.