A rule preventing patients of different sexes from being treated on the same ward was broken more than 150 times at the University Hospitals of Bristol and Weston last year, new figures show.
It comes as the total number of breaches across England has soared in recent years.
The Patients Associations said while the NHS faces "immense pressures", compromising patient dignity and privacy is "unacceptable".
The NHS rule, which was introduced in 2010, says hospitals must eliminate mixed-sex wards, except for in certain circumstances such as when patients choose to share wards with the opposite sex or when requiring highly specialised care.
Despite an initial reduction across England, the number of breaches steadily increased between 2014 and 2019, with the coronavirus pandemic since causing a significant upturn over the last five years.
NHS England figures show University Hospitals Bristol and Weston NHS Foundation Trust broke the rule 161 times in the 12 months to the end of November – up from 13 times in the year to November 2019, before the coronavirus pandemic.
Nationally, the number of breaches has more than doubled over the last five years, from 19,700 in the year to November 2019 to 49,100 last year, while the latest 12 months was the highest 12-month period for which there is complete data.
Data was not recorded between March 2020 and September 2021 because of the impact of the coronavirus pandemic, with a policy to fine trusts £250 per breach of the rule dropped in April 2021.
The single-sex rule applies to sleeping accommodation, which includes any area where patients are admitted on beds or trolleys even if they do not stay overnight.
Rachel Power, CEO of the Patients' Association, said: ""The alarming increase in breaches of mixed-sex accommodation is deeply concerning for patients.
"While we recognise the immense pressures currently facing the NHS, compromises to patient dignity and privacy are unacceptable and should not become the norm.
"The Government and NHS England leaders must urgently work together to prioritise dignified care, ensuring all patients are treated with the respect and privacy they deserve."
A spokesperson for NHS England said: "The NHS is committed to offering same sex accommodation and there is more to do to eliminate unjustified breaches.
"We recognise this is challenging for NHS trusts, especially at times of extreme pressures on services – and ensuring the privacy and dignity of patients remains paramount when same sex accommodation cannot be provided."